Denielle

Genesis B. Camato

IX. CSF ANALYSIS

ANALYSIS O F URINALYSIS AND BODY FLUIDS | REVIEWER

TRAUMATIC COLLECTION
1.
UNEVEN DISTRIBUTION OF BLOOD

blood from intracranial haemorrhage will be

evenly distributed throughout the three CSF
specimen tubes whereas the traumatic tap
will have the heaviest concentration tube 1,
gradually diminishing amounts in tube 2, and
tube 3; traumatic procedure

FORMATION & PHYSIOLOGY

First recognized by COTUGNO, 1974
Third major body fluid of the body
Provides nutrients to the nervous tissue to remove
metabolic wastes
Produce mechanical barrier to cushion brain and
spinal cord against trauma
MENINGESthose that lined the brain and spinal cord
THREE LAYERS:

Dura mater

Arachnoid mater

Pia mater
SUBARACHNOID SPACE- where CSF flows; located between
arachnoid & pia mater

Approximately 20mL /hr in choroid plexuses &

reabsorbed by the arachnoid villi to maintain total
volume of 140-170 mL in adults & 10-60mL in
neonates
BLOOD-BRAIN BARRIER- used to represent the control
and filtration of blood components to the CSF and then to
the brain

2.

TUBERCULAR MENINGITIS- classic web-like pellicle is seen

after overnight refrigeration of the fluid
3.

SPECIMEN COLLECTION & HANDLING

CSF routinely collected by LUMBAR PUNCTURE
between third, fourth, fifth lumbar vertebrae

Specimen is usually collected in three sterile tubes

labelled 1, 2, 3 in the order to which they are
withdrawn
TUBE 1- CHEMICAL & SEROLOGICAL TESTS
TUBE 2 MICROBIOLOGY
TUBE 3- CELL COUNT- least likely to contain cells
introduced by spinal tap procedure (other methods:
Systernal and Ventricular puncture)
If specimen cannot be performed on STAT basis; specimens
should be maintained in the ffg manner:

Hematology tubes are refrigerated

Microbiology tubes remain at room temperature

Chemistry and Serology tubes are frozen

XANTHOCHROMIC SUPERNATANT

results of blood that has been present longer

than that introduced by the traumatic tap;
care should be taken because a very recent
haemorrhage would produce a clear
supernatant & introduction of serum protein
from traumatic tap could also cause the fluid
to appear xanthochromic.

Additional tests for differentiation: microscopic examination &

D-dimer test
m Microscopic: macrophages ingesting RBCS
(erythrophagocytosis or hemosiderin granules)=
intracranial hemorrhage
m

D-Dimer: detection of fibrin degradation product by

Dimer, latex aggln immunoassay indicates formation
of fibrin at a haemorrhage site

CELL COUNT

APPEARANCE
Normally crystal clear CSF

Crystal Clear, Cloudy/ Turbid, Milky, Xanthochromic,

hemolyzed/ Bloody

XANTHOCHROMIA

term used to described CSF supernatant that

is pink, orange or yellow- pink (very slight
amount of oxyhemoglobin) to orange (heavy
hemolysis) to yellow (conversion of
oxyhemoglobin to unconjugated bilirubin

Other causes of xanthochromia: elevated serum

bilirubin, presence of pigment carotene, increase
protein concentrations and melanoma pigment

Xanthochromia is due to immature liver function

seen commonly in infants particularly those
premature

CLOT FORMATION

traumatic tap may form clots owing to the

introduction of plasma fibrinogen into
specimen; intracranial haemorrhage will not
contain fibrinogen to clot

Routinely performed on CSF specimens is the WBC

count
RBC counts are usually determined only when a
traumatic tap has occurred and a correction for
WBC count is needed
Cell count should be performed immediately because
WBCs (particularly the granulocytes) and RBCs will
begin to lyse within 1 hour with 40% of leukocytes
disintegrating after 2 hours

Denielle Genesis B. Camato

IX. CSF ANALYSIS

ANALYSIS O F URINALYSIS AND BODY FLUIDS | REVIEWER

METHODOLOGY

Normal adult CSF contains 0-5 WBC/uL; higher in

children (as many as 30 mononuclear cells/uL)
consider as normal in newborns

Improved Neubauer counting chamber is

traditionally used

Calculation formula:

CORRECTIONS for CONTAMINATIONS

WBC (added)= WBC (blood) X RBC (CSF)
RBC (blood)

Number of cells counted x dilution =cells.uL

Number of squares x volume of 1 square

This formula can be used in both diluted and

undiluted specimens

CYTOCENTRIFUGATION

As little as 0.1mL of CSF combined with one drop of

30% albumin produces an adequate cell yield when
processed with the cytocentrifuged.

Addition of albumin increases the cell yield and

decrease the cellular distortion frequently seen on
cytocentrifuged specimens

TOTAL CELL COUNT

Clear specimens may be counted undiluted, provided
no overlapping of cells is seen during the microscopic
examination

When dilutions are required, calibrated automatic

pipettes are used

Dilutions for total cell counts are made with normal

saline mixed by inversion and loaded in the
hemocytometer by a Pasteur pipette

Cells are counted in the four corner square on both

sides of the haemocytometer
Clarity
Dilution
Amount of
Amount of
Sample
Diluent

Slightly hazy

1 : 10

30L

270 L

Hazy

1 : 20
1 : 100

30L
30L

570 L
2970 L

1 : 200

30L

5970 L

0.1 mL of a
1:100 dilution

9.9 mL

1 : 10,000

Slightly
cloudy
Slightly
bloody
Cloudy
Bloody
Turbid

CSF CELLULAR CONSTIUTENTS

Adults have usually predominance of lymphocytes to
monocytes (70:30) ratio whereas monocytes are
more prevalent in children.

Pleocytosis- increased numbers of these normal

cells is considered abnormal, as the finding of
immature leukocytes, eosinophils, plasma cells and
macrophages, increased tissue cells & malignant cells

High WBC count of which the majority of the cells

are neutrophils is indicative of bacterial meningitis

High percentage of lymphocytes & monocytes

suggests: viral, tubercular, fungal, or parasitic origin

Cell forms differing those found in blood include

macrophages, choroid plexus, and ependymal cells,
spindle-shaped cells, and malignant cells

WHITE BLOOD CELL COUNT

Specimens requiring dilution can be diluted,

substituting 3% acetic acid to lyse the RBCs
Addition of methylene blue to the diluting fluid will
stain the WBCs providing better differentiation
To prepare a clear specimen that does not require
dilution for counting, place four drops of mixed
specimen in a clean tube.
Rinse a Pasteur pipette with glacial acetic acid,
draining thoroughly and draw the four drops of CSF
into the rinse pipette.
Allow the pipette to sit for 1 minute, mix the solution
in the pipette, discard first drop and load the
hemocytometer
2

When peripheral blood RBC and WBC counts are in the

normal range, many laboratories choose to simply
subtract 1 WBC for every 700 RBCs present in the
CSF
Differential count should be performed on a stained
smear and not from the cells in the counting
chamber
When performing diff count, 100 cells should be
counted, classified, and reported in terms of
percentage
If the cell count is low and finding 100 cells is not
possible, report only the numbers of the cell types
seen

Denielle Genesis B. Camato

IX. CSF ANALYSIS

ANALYSIS O F URINALYSIS AND BODY FLUIDS | REVIEWER

CSF CELLULAR CONSTITUENTS

Nucleated Red Blood Cells (NRBCs)- result of bone
marrow contamination during spinal tap
Eosinophils- Parasitic infection, fungal infection,
introduction of foreign material in CNS
Eg: Rat Lung Worm (Angiostrongylus cantonesis)
Entamoeba histolytica- causing amebic
encephalitis
N. Fowleri, Acanthamoeba
Macrophages- appear 2-4 hours after RBCs enter
the CSF; indicative of previous haemorrhage
Choroidal Cells- from epithelial lining of the choroid
plexuses
Ependymal Cells- from linings of ventricles and neural
canal
Spindle-shaped cells- from lining cells of arachnoid
Malignant cells of hematologic origin (blasts from
leukaemia, lymphoma cells)
Malignant cells of non-hematologic origin (metastatic
carcinoma cells) eg: breast cancer

CSF - PROTEIN

Most frequently performed chemical test on

CSF; Normally between 15-45 mg/dL

CSF protein is <1% compared to the total

serum protein

ALBUMIN is the major protein in the CSF

PRE-ALBUMIN (Transthyretin) second

most predominant protein in the CSF

IgM, Fibrinogen, Beta-Lipoprotein are not

found in normal CSF

Tau transferrin is found only in CSF; not

found in blood

Elevated total protein values are most

frequently seen in pathologic conditions.
Abnormally low values are most frequently
seen in pathologic conditions

Methods of CSF protein measurement:

Turbidimetric Mtd (eg: SSA/TCA) Dye-binding
Method (BCG/BCP)

CSF/Serum-Albumin index is used to assess

the integrity of the blood brain barrier

IgG index used to assess if there is

intrathecal IgG production within the CSF

OLIGOCLONAL Bands- for assessment of

Multiple Sclerosis; these band are also present
in disorders such as encephalitis, neurosyphilis,
Guillain-Barre syndrome and neoplastic
syndrome

Myelin-Basic Protein is indicative of recent

destruction of myelin sheath that protects
the axon of neurons (demyelination) can be
used to monitor the course of multiple
sclerosis

CSF - LACTATE

Levels greater than 35 mg/dL indicate bacterial

meningitis thus increase in lactate

Viral Meningitis- produces lactate levels lower

than 25 mg/dL thus decrease in lactate

Elevated in destruction of CNS tissue owing to

oxygen deprivation

Falsely elevated results: bloody/hemolyzed/

xanthochromic specimen

CSF - GLUTAMINE

produced from ammonia and alphaketoglutarate by the brain cells

serves to remove toxic waste product ammonia

from the CNS

Normal levels of glutamine is between 8-18 mg/dL

Elevated levels are associated with liver

disorders/ hepatic coma & Reyes Syndrome

CSF ANALYSIS - MICROBIOLOGIC EXAMINATION

Used to identify the cause of meningitis

CSF CULTURE is confirmatory test

Gram stain, acid fast stain, India ink and latex

agglutination test serve for preliminary diagnosis

India Ink for Cryptococcus neoformans; look

for capsules (fungal meningitis)

Most frequent agents include: Streptococcus

pneumonia, Haemophilus influenza, Escherichia coli,
Strep agalactiae & Neisseria meningitis (all of
these are encapsulated organisms)
CSF ANALYSIS - SEROLOGIC TESTS

For assessment of neurosyphilis using VDRL or

FTS-Abs (Treponema pallidum)
Formation and Composition of CSF

Blood brain barrier maintains the relative homeostasis of CNS

environment by tightly regulating the concentration of substances by
specific transport systems for H+, K+, Ca2+, Mg2+, HCO3-.

Glucose, urea and creatinine diffuse freely between blood and the CSF.

Proteins cross freely by passive diffusion along the concentration

gradient and is also influenced by molecular weight.
Composition of Normal CSF
Protein
15 - 45 mg/dL
Glucose
50 - 80 mg/dL
Urea
6.0 - 16 mg/dL
Uric acid
0.5 - 3.0 mg/dL
Creatinine
0.6 - 1.2 mg/dL
Cholesterol
0.2 - 0.6 mg/dL
Ammonia
10 35 g/dL
Sodium
135 150 mEq/L
Potassium
2.6 3.0 mEq/L
Chloride
115 130 mEq/L
Magnesium
2.4 3.0 mEq/L
Cells
0 5 Lymph/L
3

CSF GLUCOSE

CSF glucose is 60-70% that of plasma glucose

Eg: Viral Meningitis slightly decrease glucose

Bacterial Meningitis decrease glucose

Blood glucose should be drawn in conjunction with

CSF values; blood glucose should be drawn 2
hours before the spinal tap

CSF ANALYSIS CHEMICAL EXAMINATION